Compact, high-resolution fluorescence and brightfield microscope and methods of use

ABSTRACT

The present invention provides a compact, inexpensive fluorescence microscope capable of high-resolution imaging with high light throughput suitable for use in both laboratory and field environments, and methods of use. A simple and inexpensive fluorescence microscope allows health care workers to perform various medical assays at the point of care instead of having to collect and transport biological samples to distant labs, and subsequently return the results to the patient. The microscope of the present invention is also useful for educational use and field use, and other uses as well.

The present application claims benefit of priority to U.S. ProvisionalApplication No. 61/458,696, filed on Nov. 30, 2010, entitle “Compact,High-Resolution Fluorescence Microscope,” which is incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention generally relates to a digital inverted or uprightfluorescence microscope having excitation, emission, and imaging opticsand sensor integrated into a short light path in a single compactassembly for high resolution, and using a CMOS sensor for imageacquisition and control of image display on an electronic device withoutneed for an ocular assembly.

BACKGROUND

Fluorescence and brightfield microscopes have become indispensable toolsfor science and medicine. Traditional laboratory microscopes with photocapabilities needed for research and medical diagnostics are large,cumbersome, fragile, and expensive to the extent that they are pricedout of reach for many potential users, including science teachers.Accordingly, many researchers, clinicians, and educators lack access tomicroscopes for their work. Furthermore, many microscopes arecomplicated to use and maintain, which hinders their use in manyapplications.

Schools struggle with modern conventional microscopes. They are foreignand intimidating to many students. Their complicated operation resultsin frequent difficulties such as misalignment, inappropriate interoculardistances, incorrect condenser focus, and damaged or dirty objectivelenses, in some cases due to the physical inaccessibility of these partsto the user. In many classrooms, the number of students easily exceedsthe ability of a teacher to assist and verify what the students are orare not seeing. Because of these difficulties, teachers have resorted tousing sophisticated microscope simulations since microscopy is such animportant component of the modern curriculum(http://virtualurchin.stanford.edu/microtutorial.htm).

Many modern disease diagnostic assays utilize fluorescence, whetherintrinsic to the sample, provided by the binding of a fluorescentmolecule or an antibody labeled with a fluorescent moiety specific to adisease epitope, indirect immunofluorescence, or in situ hybridizationof a fluorescently labeled nucleic acid sequence to a genetic marker ofdisease, among other diagnostic approaches. These diagnostic assays arelimited in their availability to parts of the world where thedeleterious impact of these diseases is greatest, due in part to theoperational complexity and expense of fluorescence microscopes. Malariainfects an estimated 225 million people worldwide, yet many more casesmay remain undiagnosed. During its lifecycle, the parasite dwells withinthe confines of red blood cells where it can be observed in a bloodsmear with suitable contrast enhancement or, because red cells containno chromosomes, with a simple membrane-permeant fluorescent dye thatintercalates and stains DNA. Yet due to the expense and cumbersomenature of modern fluorescence microscopes, these simple diagnostic testsare not being performed at locations where they are needed.

We describe a highly economical and compact inverted fluorescencemicroscope system, incorporating a brightfield and oblique transmissionimaging mode and having a simple, yet robust design, with broadapplications in research, science education, and point-of-care medicineto address these unmet needs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a rear cross-sectional view of the layout of a compacthigh-resolution fluorescence microscope of the present invention.

FIG. 1B depicts a top cross-sectional view of a compact high-resolutionfluorescence microscope of the present invention.

FIG. 2A depicts a cross-sectional view of an optical imager assembly ofa compact high-resolution fluorescence microscope of the presentinvention.

FIG. 2B depicts another cross sectional view of an optical imagerassembly of a compact high-resolution fluorescence microscope of thepresent invention.

FIG. 3A depicts a cross-sectional view of a focusing assembly of acompact high-resolution fluorescence microscope of the presentinvention.

FIG. 3B depicts another cross-sectional view of a focusing assembly of acompact high-resolution fluorescence microscope of the presentinvention.

FIG. 3C depicts cross-sectional view of a focusing assembly of a compacthigh-resolution fluorescence microscope of the present invention.

FIG. 4 is a photograph of an assembled compact high-resolutionfluorescence microscope of the present invention.

FIG. 5A depicts an electrical subsystem of a compact high-resolutionfluorescence microscope of the present invention.

FIG. 5B depicts another electrical subsystem of a compacthigh-resolution fluorescence microscope of the present invention.

FIG. 6 depicts a display of a host computer running the microscopecontrol program whiles acquiring images cells.

FIG. 7 depicts an image of fixed Paramecium tetraurelia made using acompact high-resolution fluorescence microscope of the presentinvention.

FIG. 8 depicts an image of a horizontal section through a leaf of Viciafava made using a compact high-resolution fluorescence microscope of thepresent invention.

FIG. 9 depicts an image of Spirogyra crassa made using a compacthigh-resolution fluorescence microscope of the present invention.

FIG. 10 depicts an image of bovine pulmonary arterial endothelial cellsmade using a compact high-resolution fluorescence microscope of thepresent invention.

FIG. 11 depicts an image of bovine pulmonary arterial endothelial cellsmade using a compact high-resolution fluorescence microscope of thepresent invention.

FIG. 12 depicts an image of transgenic nematodes made using a compacthigh-resolution fluorescence microscope of the present invention.

FIG. 13 depicts an image of transgenic nematodes made using a compacthigh-resolution fluorescence microscope of the present invention.

FIG. 14 depicts a brightfield image of a transverse section of thedermal layer of human skin containing a nevus or mole from a biopsy madeusing a compact high-resolution fluorescence microscope of the presentinvention.

FIG. 15 depicts a fluorescent image of a transverse section of thedermal layer of human skin containing a nevus or mole from a biopsy madeusing a compact high-resolution fluorescence microscope of the presentinvention.

FIG. 16 depicts an image of an unstained section of normal human smallintestine showing auto-fluorescence made using a compact high-resolutionfluorescence microscope of the present invention.

FIG. 17 depicts an image of a section from a small intestinal tumorobtained from a human patient made using a compact high-resolutionfluorescence microscope of the present invention.

FIG. 18 depicts an image of fetal telencephalon-derived human neuralstem cells obtained by time-lapse image acquisition.

FIG. 19 depicts an image of fetal telencephalon-derived human neuralstem cells obtained by time-lapse image acquisition.

SUMMARY

The present invention recognizes the need for a compact, inexpensivefluorescence microscope capable of high-resolution imaging with highlight throughput suitable for use in laboratory and field environments.

A first aspect of the present invention is an inverted fluorescencemicroscope.

A second aspect of the present invention is a method of using aninverted fluorescence microscope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein are well known and commonly employed in the art. Where aterm is provided in the singular, the inventors also contemplate theplural of that term. The nomenclature used herein and the proceduresdescribed below are those well known and commonly employed in the art.

Introduction

The present invention recognizes the need for a compact, inexpensivemicroscope capable of high-resolution imaging with high light throughputsuitable for use in laboratory and field environments.

As a non-limiting introduction to the breath of the present invention,the present invention includes several general and useful aspects,including:

-   -   1) an inverted fluorescence microscope including a stage for        placing a sample for observation and a compact, integrated        epifluorescence illumination and detection system; and    -   2) a method of using an inverted fluorescence microscope of the        present invention.

These aspects of the present invention, as well as others describedherein, can be achieved by using the methods, articles of manufactureand compositions of the matter described herein. To gain a fullappreciation of the scope of the present invention, it will be furtherrecognized that various aspects of the present invention can be combinedto make desirable embodiments of the present invention.

I. An Inverted Fluorescence Microscope

The present invention includes an inverted fluorescence microscopeincluding a stage for placing a sample for observation and a compact,integrated epifluorescence illumination and detection system.

In the present invention, a single-assembly light path directsexcitation illumination from a miniaturized source to a sample andfocuses the resulting fluorescence image onto an integrated CMOS(complementary metal-oxide-semiconductor) active-pixel sensor. Imagedata is transmitted from the sensor to a host computer or otherelectronic device by a standard universal serial bus (USB) forobservation on a display screen. The present invention is unique in itsdesign, unlike existing fluorescence microscope, enabling broad utilityin research, diagnostics, and education.

The low light-loss imaging assembly includes one or more of thefollowing:

-   -   1) an objective lens system for focusing the image of the sample        on the sensor;    -   2) an excitation light source;    -   3) filters for selecting one or more wavebands of excitation        light, for directing said light to the sample, and for selecting        one or more wavebands of light emitted by the sample;    -   4) tube or projection lenses; and    -   5) an electronic imaging sensor.

The sensor is arranged in a compact manner in a single assembly withoutreflecting mirrors to decrease internal reflection, maximize lightthroughput, and enable formation of a high-resolution image. Theintegrated CMOS sensor has analog and digital signal and imageprocessing functions integrated with the active pixel array onto asingle fabricated semiconductor wafer, enabling facile control of imageacquisition and post-acquisition processing under computer directionthrough a fast, standard communication bus, such as USB. The imagingassembly is mounted to the sample stage via a focusing mechanism alsomounted to an enclosure or chassis on which said stage is mounted, suchthat an optical axis is mechanically defined and pinned at multiplefulcrum points, to provide exceptional stability of the image on thesensor and resistance to vibration and other mechanical shock.

Wide-field fluorescence microscopes in common use obtain high resolutionimages with long and complex light paths in which excitation lightdirected to the sample and light emitted or scattered by the sample aredirected to their intended targets by the use of multiple reflecting orrefracting surfaces (for example, U.S. Pat. No. 7,639,420). In addition,multiple compound lenses containing numerous elements to correctspherical and chromatic aberrations are used to obtain high-resolutionimages of the sample. However, light intensities in the sample attenuatewith the inverse square of the distance, and each reflecting orrefracting surface in an optical path decreases light throughput in theoptical system, such that fluorescent samples emitting weak lightintensities may not be detected because insufficient light reaches thedetector, whether the eye or a camera. The present invention surmountsthis shortcoming by focusing on the more important optical elements ofthe fluorescence microscope and placing them on the shortest, directoptical path between sample and sensor. Optionally removing from afluorescence microscope the optical interface to an eye—the oculareyepiece—enables the construction of vastly smaller, simpler, cheaper,more robust, and more sensitive microscopes.

Another aspect addressed by the invention is the electronic sensor usedto record the fluorescence image of the sample. The literature reportsthat the need for eyepieces can be reduced by the use of a digitalimager (see, for example, U.S. Pat. No. 7,599,122) such as CCD (chargedcoupled device) or standard CMOS pixel arrays. CCD and standard CMOScameras have relatively large power requirements for control circuitry,external to the pixel array, preferably to time image acquisition, clocklight-induced pixel charge out of the array, convert the analog voltagesignal of each pixel to a digital value, and format the imaging arraydata for computer displays and other camera functions. These functions,preferable for the operation of the array as a camera, are bulky andresult in a relatively large camera container, because they cannotreasonably be integrated onto the semiconductor wafer on which the CCDor standard CMOS array is fabricated. This container is typicallymounted into the microscope platform, resulting in the need for steeringthe image to the camera port, requiring additional optical lensingelements to achieve a focused image. In addition, the external circuitrygenerates significant heat, which increases the dark thermal noise ofthe CCD or CMOS array. Even when this heat is dissipated by a radiatoror fan, achievement of an acceptable noise level often requiresadditional cooling of the imaging array. Furthermore, CCD cameras have atendency to bloom or create streaking artifacts if too much charge isdeposited in their pixels. Larger pixels of the CCD, 10 to 13 μm lineardimension, offer greater dynamic range, but come at greater cost ofsilicon, and also require higher magnification of the optical system. Incontrast, integrated CMOS imaging detectors are not prone to bloomingbecause they do not employ a “bucket brigade” algorithm to transfercharge to the readout amplifier. Instead, each active pixel element isread out through its own individual amplifier. Their pixels can be madesmaller, to the range of 2 to 3 μm, and thus lower optical magnificationis required to achieve higher image magnifications. The decreased pixelsize decreases the noise charge accumulated in the dark. Moreover, aside channel FET, integrated into each active pixel element, removesthermal charge accumulating in said element during the dark, when animage is not being captured, to keep the noise low, thus eliminating theneed for cooling (see, for example, U.S. Pat. No. 7,102,672). Thepresent invention obviates these difficulties by the use of anintegrated CMOS instead of standard CMOS imager. The integrated CMOSimaging sensor has a high dynamic range active pixel array in whichimage control functionality and dark charge control are integratedmonolithically on the imaging array wafer, resulting in a very small,fully functional image sensor package, in other words, a complete cameraon a chip.

Another aspect addressed by the invention is means of control of theelectronic sensor and, hence the resulting image. External clocking andtiming, acquisition control, digital signal processing, and imageformatting functions in CCD or simple CMOS cameras cannot reasonably beintegrated onto the pixel array semiconductor, and instead areoptionally present as separate device elements connected to the array byexternal connectors. This invention takes advantage of the placement ofclocking and timing, image acquisition and formatting, digital signalprocessing, and system control functions into circuits within thephysical package of the integrated CMOS imaging array, such that powerconsumption is greatly reduced, the physical size of the container isdrastically decreased, and image sensor functionality is increased,enabling greater control of the resulting fluorescence image. Theinvention realizes these advantages of the integrated CMOS imagingsensor by making further use of the USB data communication standard forsystem control at the register level on the integrated CMOS sensor bythe host computer, and for formatting and transferring imaging data tosame for display. This allows direct control of functions determiningimage quality, such as signal gain, exposure time, output image framerate, as well as switching to automated control modes, in which internalsystem control algorithms are used to set gain and exposure to enablemapping the intensity range of the image to the dynamic range of theactive pixel array.

The technical challenge of the invention may be attained in accordancewith the invention by providing a microscope comprising a horizontalstage for placement of a sample to be observed through an observationhole, with the stage serving as the top surface of the microscopeenclosure. The essential elements of the fluorescence microscope aremounted on a single fixture, the imager tube, located below theobservation hole inside the microscope container. These elements includean objective lens mounted to the top surface of the fixture such thatits optical axis is oriented perpendicular to the plane of the stage andits field of view is centered at the center of the observation hole, adichroic filter mounted below the objective at a 45° angle to theoptical axis, and an illumination tube mounted to the fixture in aposition so as to direct light toward the dichroic filter in a directionperpendicular to the optical axis. The illumination tube contains alight source, which in a preferred embodiment is a light-emitting diode(LED) mounted to a miniature power-conditioning circuit, a series ofcondenser lenses, which allows control of vergence of the excitationlight on the sample, and an excitation barrier filter, which selects oneor more wavebands of light in ranges of wavelengths less than the cut-ontransmission wavelength of the dichroic filter. The axis of theilluminating light is oriented at a 90° angle with respect to theoptical axis defined by the objective, such that the dichroic filterreflects the selected excitation light to the sample on the stage at thecenter of the field of view. The emission barrier, multiple-barrier, orlong-pass filter is mounted to the fixture below the dichroic mirror toselect desired wavelengths of fluorescence emitted by the sample forobservation. A series of projection lenses mounted in tandem below theemission filter serve to focus this selected fluorescence on the imagesensor.

In a preferred embodiment of the invention, the imager tube assembly ismounted to a focusing mechanism that is in turn mounted to the chassisof the container. The image of the sample is focused on the sensor byadjusting the vertical distance between the objective lens entrancepupil and the sample. The chassis mounting resists vibrations thatdegrade image quality and improves resolution of the image.

In an alternate embodiment, the focusing mechanism can control thestage. The focusing mechanism can be mechanically connected to a humaninterface device such as a knob, or it can use an electromechanicalmechanism controlled through an electronic user interface.

In a preferred embodiment, the image sensor is a high-density array ofwide dynamic range active CMOS pixels monolithic with a set of analogand digital control and processing circuitry embedded on the samesemiconductor wafer. The integrated CMOS sensor enables the imagedetector to be compact and economical without circuit boards andprocessors located on platforms external to the sensor wafer, whichgreatly improves functionality and decreases power requirements, whichdecreases the dark noise of the imaging pixels and enables detection oflow levels of light.

In a further aspect of this preferred embodiment, the control andprocessing circuitry of the image sensor is interfaced to a computer bya Universal Serial Bus. The bus allows communication between the sensorand host computer. The image sensor is programmed by the host computerto acquire and format the image data in a manner compatible for displayon the computer monitor by this bus. The standard communication formatenables the sensor to be programmed to control image acquisition by theintegrated CMOS active pixel array, and to control processing of theacquired image under direction by the user. Other standard computerinterfaces, such as Ethernet, can embody the same functionality.

In a further aspect of the preferred embodiment, a manual or automatedx-y caliper is mounted to the sample stage, to enable the sample to befixed in horizontal dimension for clamping the sample in the field ofview and to enable controlled surveying of the sample at specificlocations.

In a further aspect of the preferred embodiment, a brightfieldillumination source is attached to the external surface of the containerto allow transmission illumination of the sample for location of regionsof interest to be placed within the field of view, and to enableadjustment of the plane of focus within the sample.

In a further aspect of the preferred embodiment, the USB connectionprovides power from the host computer to the sensor, the fluorescencelight source, the external brightfield light source, and other usefulparts of the microscope.

This arrangement reduces light losses by elimination of reflectiveelements normally used to steer light in other microscope designs. Themicroscope does not use oculars or eyepieces for focusing orobservation, but instead relies on an integrated CMOS active pixelimaging array to produce a digital image displayed in real time on acomputer. The imaging array controls image acquisition on the sensorwithout the need for external circuitry, which enables significantprocessing of the image before transmission to the computer. Themicroscope eliminates the need for refractive index matching oradjustment typically needed for high-resolution high-magnificationimaging.

FIG. 1A and FIG. 1B show respectively a rear and top externalperspective of the layout of a preferred embodiment of the invertedmicroscope. The container comprises a sample stage (1) sitting atop anenclosure defined by the main unit case 2 a and rear cover 2 b. Thestage (1) is an anodized aluminum or other metal or stiff materialfabricated to an end-to-end flatness of ≦0.06 mm. The bottom half of thestage is fabricated in a manner to create an understage that extendsbelow the top surface of the enclosure to rest within the footprint ofthe enclosure. The edges of the top surface of the stage extendingbeyond the understage may extend beyond the horizontal footprint of thecontainer. This overhang enables large samples, such as cell and tissueculture flasks, to be supported in a stable manner on the stage. Theunderstage portion of stage (1) is machined to dimensions identical tothe horizontal footprint defined by the main unit case 2 a and rearcover 2 b. The stage (1) is attached to the enclosure by screws throughholes in main unit case 2 a and rear cover 2 b where they overlap therim of the understage portion. These attachments mount stage (1) firmlyto the enclosure and minimize vibrations and bending and other motionsthat could cause instability of the image on the sensor.

Base (3) is a metal or other stiff material plate machined tosubstantial flatness, with plastic or rubber feet to enable thecontainer to sit flat on a table, bench, or other working surface. Theenclosure is mounted to base (3) by screws at the bottom of thecontainer in a manner such that the top surface of stage (1) ishorizontal within ±0.2 mm. These arrangements of the external layout ofthe inverted microscope provide a stable platform for opticalobservation of the sample with minimum vibration

Electrical connections to the microscope are made by connectors mountedin holes through the enclosure. These connections include the USBconnection for the sensor (4), the connection from the power supply forthe fluorescence illumination (5), the connection for the brightfieldillumination source (6), and connections for other functions.Connections 5 and 6 may be used when it is not desired to obtain powerfor the fluorescence and brightfield illumination sources from the USBconnection. In addition, switches for brightfield and fluorescenceilluminators are mounted on the enclosure to enable independentactuation of each mode of illumination.

The outer surface of the enclosure, main unit case 2 a and rear cover 2b, may be painted or otherwise covered with a material having pleasingappearance, such that brands or other labels may be attached to providethe user with information of interest or need.

Since other modifications and changes varied to fit particular operatingrequirements and environments will be apparent to those skilled in theart, the invention is not considered limited to the example chosen forpurposes of disclosure, and covers all changes and modifications whichdo not constitute departures from the true spirit and scope of thisinvention.

Internal Layout of Microscope Components

Referring again to FIG. 1A and FIG. 1B, the general internal layout ofcomponents in a preferred embodiment of the inverted microscope isshown. In this preferred embodiment, the focusing assembly (7) ismounted to the bottom of the stage at two or more distinct horizontallocations to establish a horizontal axis for focusing motions. As isdescribed further in “Focusing assembly”, this enables provision of astable mechanical axis for lifting and lowering the optical imagingassembly (8) in the motions required for focusing the image of thesample on the sensor. In the preferred embodiment, the optical imagingassembly (8) is mounted to the focusing assembly (7) to enable focusingwhile preserving mechanical stability.

Optical Imager Assembly: Imager Tube and Objective

Referring to FIG. 2A and FIG. 2B, the optical imager assembly is builtwith an imager tube (9), to which other components of the assembly aremounted. The objective lens (10) is mounted to the hole through the topof the image tube (9) with sides of the hole machined to the same screwthread size as on the base of the objective lens housing to secure atight fit. The objective lens is positioned below the circularobservation hole cut through the stage (1).

In the preferred embodiment, a wide variety of objective lenses may beused. One optional criterion is that the lenses are fabricated fromnon-fluorescent materials, such as fused silica, quartz, or calcite, todecrease the intrinsic autofluorescence of the microscope. They may becompound lenses designed with finite tube lengths, and the dimensions ofthe optical imaging assembly are designed to accommodate 160 mm tubelengths, the industrial standard. Alternatively, the lenses may beinfinity-corrected assemblies, in which case projection lenses areinserted in the imager tube to enable the sample image to be focused onthe image sensor. Many different objective lenses manufactured withstandard microscope mounting screw threads can be used in the invertedmicroscope. Representative, but not exclusive example of such lensesinclude the Plan 40× 0.65 NA 0.5 mm working distance, Plan 20× 0.4 NA7.3 mm working distance, and Plan 10× 0.25 NA 7.5 mm working distanceobjectives (Meiji Techno, Japan), although many other lenses, includingvideo camera lenses and have been used and found suitable.

In an alternative embodiment, further cost and size reductions arepossible by using simple infinity-corrected lenses designed for webcamsand low-magnification applications. Because of the small (2-5 micron)pixel dimensions of integrated CMOS imaging sensors, opticalmagnification greater than 5-10× will exceed the diffraction limit andadd no further resolution to the image. Such low magnifications may beachieved with simpler lenses than microscope objectives. Lowmagnification lenses may perform better in the microscope by invertingthem front-to-back.

In the preferred embodiment, the diameter of the observation hole is atleast 1 mm greater than the outermost diameter of the objective lenshousing to enable unimpeded motion of the objective housing duringfocusing through the stage 1, and to enable removal of an objective lenswhen changing said lenses.

Optical Imager Assembly: Fluorescence Filters and Epi-Illumination

Below the objective lens, a hole is machined through the side of imagertube to accept the filter tube (11). The sides of this hole are smoothsuch that the filter tube is correctly positioned and mounted to thetube by the tightness of the press fit and by a retaining screw. Thefilter tube (11) is centered in the hole by a surrounding o-ring. Theend of the filter tube sitting in the space below the objective ismachined to a 45° angle with respect to the optical axis defined by thecentral axis of the objective (axis A in FIG. 2B) and the centrallongitudinal axis of the filter tube (11). The mounting arrangementdescribed enables this end to be positioned in this prescribed manner.The dichroic filter (12) is mounted to this 45° angle surface by the useof two retaining screws with washers in two holes flanking the outeredges of the dichroic filter (12). Flock paper inserted on the top andbottom surfaces of the dichroic filter (12) where the washers contactthe filter are used to enable a compression mounting of the filterwithout damage to the glass. The dichroic filter is selected so as toallow the desired wavelengths of light emanating from theepi-illumination light source to be reflected to the sample through theobjective lens so as to excite the fluorophores in the sample, yet alsoto allow the wavelengths of light emitted by said fluorophores to betransmitted through the dichroic filter.

The end of the filter tube (11) protruding from the imager tube (9) ismachined to accommodate the epi-illumination system (13). Theepi-illumination system is also configured as a cylindrical tube that isaccepted into a counter-bored hole in the filter tube and centered andpositioned in place with retaining screws through the filter tube. Theillumination tube holds the epi-illumination light source (14) at theinternal end of the tube. In the preferred embodiment, the light sourceis a light-emitting diode (LED), such as a LUXEON Rebel (PhilipsLumileds Lighting Co., San Jose, Calif.) with an emission spectrumproviding significant luminance at the excitation wavelength(s) of thefluorophore(s) of interest in the sample. Behind the light source is theminiaturized circuit board (15) containing the current-voltage controlapparatus for powering and gating the light source output. The circuitboard is connected to the connector for the fluorescence illumination(5) in FIG. 1A and FIG. 1B by leads that, in the preferred embodiment,are connected to a manual switch located in the main unit case 2 a forpowering the light source on and off.

The epi-illumination from the light source is collimated or focused onthe sample by reflection from the dichroic mirror and passing throughthe objective lens. To achieve greater control of the degree of vergenceof the epi-illuminating light upon the sample, a condenser opticalcircuit (16) comprising one or more lenses of the desired front and backcurvatures and intervening spacers is interposed between the lightsource and the end of the filter tube (11) internal to the opticalimager unit (9). This optical circuit is composed of lenses separated byspacers and assembled as a stack before insertion into the filter tube(11) side hole. The first element of the stack is the excitation filter(17), which is a single or multiple band pass interference filter thatis tuned to transmit the desired wavelength(s) of light present in theillumination from the light source to excite the fluorophores in thesample. The arrangement allows the condenser optical circuit (16) toabsorb undesired heat from the light source, if present, before theexcitation wavelengths are selected by the excitation filter (17).

In an alternate embodiment of the invention, the light source in theepi-illumination system 13 is a laser diode that emits one or morewavelengths of light overlapping the excitation spectrum or spectra ofthe fluorophore or fluorophores in the sample. In this embodiment, theexcitation filter (17) can be removed to increase the intensity ofepi-illumination at the sample.

In the preferred embodiment, the emission filter (18) is located belowthe dichroic filter in the imager tube (9) in a counter-bored cut-out ofthe imager tube. The emission filter (18) faces the dichroic filter andthe back end of the objective. The emission filter is selected to passoptimally the waveband(s) of light emitted by the fluorophore(s) in thesample without transmission of the excitation. Below the emission filterare projection lenses (19) inserted into a stack when infinity-correctedobjectives are used. The entire stack of projection lenses (19) andemission filter (18) are fixtured in the imager tube by retention nut(19 a). The imager tube at the emission filter-projection lens isthreaded along its length, such that when finite-tube objectives areused, and the projections lenses are not present, the nut snugly seatsthe emission filter (18) into the imager tube.

Optical Imager Assembly: Image Sensor

The image sensor (20) is arranged below the projection lens assembly inthe direction of sample side to image side to receive an image of thesample focused by the objective lens and projection lenses, and convertsthe optical signal into an electronic signal. In a preferred embodiment,the image sensor (20) is mounted to a circuit board that is mounted tothe bottom surface of the imager tube (9). In another aspect of thepreferred embodiment, the image sensor (20) is an integrated highperformance, low-voltage CMOS imaging active pixel array in whichfunctional elements for timing and control of image acquisition andreadout of the resulting electronic image are embedded monolithicallywithin the circuitry of the semiconductor wafer on which the imagingpixel array is created. These functional elements include rowaddressing, column sample and hold, an amplifier for each pixel withgain control, analog-to-digital conversion, black level calibration,digital signal processing, image formatting for computer displaycompatibility, image output, registers for system control, registers forinterface control, and an internal timing generator such as aphase-locked loop. The circuit board on which the wafer is mountedcontains circuit elements for input/output control and voltageregulation in a compact package that seats within the diameter of theimager tube (9). Suitable image sensors include CMOS imager chips usedin digital cameras, webcams, and cellular telephones, such as the 9712,9715, and 6552 (Omnivision Technologies, Sunnyvale, Calif.) as well asothers.

In a preferred embodiment of the invention, the image sensorcommunicates with the host computer by a USB version 2.0 bus. Amultiple-lead electrical cable connects the circuit board of imagesensor 20 to a USB bridge located within the enclosure (FIG. 1A and FIG.1B). The USB bridge, in turn, is connected to the USB connector 4located on the main unit cover 2 a (FIG. 1A and FIG. 1B). The USB bus isused to configure the image sensor registers, to provide an externalclock for image acquisition and readout, to establish timing parametersnecessary for reading out the image data in a computer-compatibleformat, to encode the image data into a display format, such as displayresolution (e.g., 640×400 pixels SVGA, 1280×800 pixels WXGA format, orgreater resolution formats), to establish the rate at which formattedimage frames are read out of the sensor, to choose automatic or manualgain and exposure of image acquisition by the sensor, to set manual gainvalues or exposure times for image acquisition and processing, or othersignal and image processing functions. All or some control of the imagesensor is enacted through the USB by software running on the hostcomputer.

Focusing Assembly:

Referring to FIG. 1A and FIG. 1B and FIG. 3A, FIG. 3B, and FIG. 3C, theoptical imaging assembly (8) is mounted to the focusing assembly (7) byan interface plate (21).

Referring to FIG. 3A and FIG. 3B, the focusing knob (21 a) is mounted bya set screw to the shaft protruding through a hole drilled through themain unit case (2 a). This shaft terminates in a planetary reductiondrive (22), which is mounted by an L-bracket to the underside of thestage (1) by two screws that are tightened so as to position therotational axis of the shaft parallel to the horizontal plane of the topsurface of the stage (1) and perpendicular to the object side to imageside of the optical imaging assembly (8). Planetary reduction drive (22)is connected to shaft (23) by a compression fitting consisting of anouter rubber or other pliant material o-ring concentric with an innerplastic or other pliant material cylindrical washer having radialthickness such that when shaft (23) is pushed into the counter-boredreceptacle in the planetary reduction drive (22), a tight fit is made.This method of attaching shaft (23) to drive (22) enables the opticalimager assembly (8) to be traversed through the full range of verticaldistance enabled by focusing assembly. The flexible compressioncoupling, however, absorbs any force of collision between the front lensof the objective lens (10) with the sample to prevent damaging the frontlens, the sample, or the focusing assembly.

The opposite end of shaft (23) is inserted in a hole drilled through themounting block (24). Mounting block (24) is attached to the underside ofthe stage by at least two screws through the block. The through-hole inmounting block (24) is precision milled to have a diametrical toleranceof preferably 2 μm such that shaft (23) fits snugly, but is still ableto rotate without detectable sticking. The end of shaft (23) terminateswith a circular brass disk (25). On the side of the disk facing theoptical imager assembly, a spiral groove is milled, preferably having awidth >2 mm and a depth >3 mm, to accept a dowel pin (21 b) insertedinto the interface plate (21) and to enable three complete circularturns of the disk between maximum upward and downward positions of theoptical imaging assembly (8), which in a preferred embodiment, is adistance of at least 25 mm This allows for different heights of samplesand desired planes of focus in samples on the stage 1. The groove may bebox ended or may have a radius, but in the preferred embodiment, thelength of the dowel pin (21 b) protrusion into the groove is less thanthe groove depth such that the primary contact between dowel pin (21 b)and disk is along the side wall of the spiral. This enables the up anddown focusing traverse to be smooth. In a preferred embodiment, thediameter of the disk is >25 mm and the pitch of the spiral isapproximately 3 mm, such that three complete turns of the spiraltraverse a full length of 75 mm.

Interface plate (21) is attached to the mounting block (24) through alinear bearing slide (26). The outer sleeve of the slide (26) is facedto the mounting block (24) and attached to the block by four sockethead-cap screws inserted through the opposite side of the block suchthat only the threaded portions of the screws extend beyond the mountingblock. The accepting holes for these screws in the linear slide (25)sleeve are located near the corners of the sleeve. The inner slide ofthe linear motion slide (26) is attached to the interface plate (21) bysocket head cap screws inserted into holes with countersunk cutouts forthe head caps from the mounting block (24) side with threaded holesdrilled into the inner slide. The linear slide (26) is located near thecenter of the interface plate (21) to provide a balanced mechanicalsupport for the interface plate (21) and its attached optical imagerassembly (8), as the imager assembly (8) is lifted and lowered by thedowel pin (21 b) via the interface plate (21).

This focusing assembly (7) and its method of attachment to the opticalimager assembly (8) provide mechanical support for the main elements ofthe microscope during focusing motions. The entire weight of the opticalimager assembly (8) is borne by focusing assembly (7). The focusingassembly is mounted to the underside of the stage at two distinctlocations, the L-bracket attachment for planetary reduction drive (22),and the mounting block (24) that supports the optical imager assembly(8) at the dowel pin and restricts its motion to a vertical direction bythe linear motion slide (26). The two-location attachment of thefocusing drive train—comprising the focusing knob (21 a) and its shaft,planetary reduction drive (22) and its shaft (23) terminating at thespiral disk (25) provides two fulcrums for establishing and maintainingthe horizontal mechanical axis for rotational motion of the focusingassembly. This requires machining of mounting block (24) such that thecenter of the through-hole for shaft (23) can be placed at a verticaldistance below the underside of stage 1 within a tolerance of 0.1 mm.The L-bracket attachment of planetary reduction drive (22) is machinedsuch that the vertical location of the corresponding center of the shaftwithin the drive can be placed at a vertical distance below theunderside of stage (1) within the same tolerance. In addition, focusingassembly (7) is assembled in a specific order to achieve the optimalorientations of the horizontal focusing axis with respect to the topsurface plane of stage (1). The circular spiral disk (25) is mounted toshaft (23) and inserted through the acceptance hole in mounting block(24), which is loosely screwed to the underside of stage (1). Theplanetary reduction drive (22) is then inserted into the acceptance slotof its L-bracket so that its shaft extends through the hole in the mainunit case 2 a, and the L-bracket is loosely attached to underside ofstage (1). Only after the flexible compression fit at the end of shaft23 is pressed into its receptacle in the planetary reduction drive (22)and shaft (23) is positioned horizontal to the underside of stage (1)and parallel to the front wall of unit case 2 a are the L-brackettightly screwed to the underside of stage (1), planetary reduction drive(22) firmly screwed to said L-bracket, and mounting block (24) tightenedto the underside of stage (1). Alignment of shaft (23) with the stage(1) underside and unit case 2 a wall are easily performed with astraight-edged ruler or other metric device. Finally, the linear bearingslide (26) is attached to interface plate (21), and both interface plate(21) and the outer sleeve of the linear bearing slide (26) are attachedto the mounting block (24). The screw-holes for these attachments arelocated at positions on the mounting block that allow easy access whenthe shaft (23) is already attached to the block. The microscope assemblyis finished by attaching the optical imager assembly (8) to theinterface plate (21) at two slightly offset locations, one directlybelow the dowel pin and one located on the opposite side of the linearbearing slide (26). These attachment locations maintain the optical axisof the optical imager assembly (8) vertical with respect to the plane ofthe stage (1) and prevent toppling.

A photograph of the assembled microscope is shown in FIG. 4.

Microscope Control:

Referring to FIG. 1A and FIG. 1B, in a preferred embodiment, electricalpower to the excitation light source is available from either anexternal power source connected to connector 5 or the USB controlconnector 4. Electrical power to the remaining subsystems is derivedfrom the 5 V USB power. Both connectors 4 and 5 terminate on anelectrical control board (4 a) that contains the electrical subsystemsof the microscope, with the exception of the excitation light source(14), and image sensor (20)

In a preferred embodiment, the microscope is controlled by a programrunning on the host computer that initially configures the on-chipcontrol registers of the integrated CMOS sensor to continuously acquireimage data and output said data in a specified format and at a specifiedimage rate to a volatile buffer in the USB interface, from which thehost continuously reads the formatted data to a buffer in the memory ofthe host computer from whence it is displayed through a graphical userinterface. In a further specification of the preferred embodiment, theprogram runs on the host in a mode in which the memory required for itsoperations is protected from being overwritten by the computer'soperating system, and assertions to the operating system from theprogram are handled with a priority enabling continuous display ofcomplete images acquired by the sensor in a manner that appears smoothand pleasing to the human eye. In a further specification of thepreferred embodiment, the host program enables the user to selectbetween modes in which the image sensor uses on-chip processing of thelight intensity values in an image to control its gain, exposure time,the rate at which it outputs complete, formatted image frames, and otherimage acquisition parameters, or uses values for said parametersselected by the user and written to the appropriate control registers ofthe sensor.

Referring to FIG. 5A and FIG. 5B, power is delivered to the microscopefrom the USB connector to the host computer. The block diagram of powerdistribution and control of the brightfield and excitation light sourcesand the image sensor is outlined as a block diagram in FIG. 5A. USBpower is delivered by the USB connector from the host computer. Currentlimitation and voltage regulation of this 5 V supply is used to powerthe brightfield led, the image sensor (20), the USB interface, and thelocal microcontroller. When external power is present, the localcontroller switches the excitation light source (14) power source fromthe 5 V USB supply to the external power source.

Control of the excitation light source power source (27) is depicted inFIG. 5B. Power is derived for the microscope from the host computer (28)via the USB connector (29). The USB connection from the host computer(28) delivers USB power (29 a) to the microscope, and enables controlcommunication (29 b) with the local microcontroller (30). In the absenceof external power supplied to the external power connector (31), thelocal controller (30) enters USB power mode for the light source (27).In USB power mode, local controller (30) closes the USB SPST powerswitch (32 a) after first opening the external power SPST switch (32 b),whose outputs are connected in common to one terminal of the lightsource (27), and sets its control output (33) high to the AND gate (34).Illumination by the light source (27) is controlled by its current,which is determined by a voltage-controlled current regulator (35) setto a desired value by a constant voltage reference (36) also connectedto USB power (29 a). The output of the constant voltage reference (36)of the USB supply is connected to the inverting input of the comparator(37) to which is connected the output of a digital-to-analog converteror DAC (38) at its non-inverting input. The DAC (38) outputs a currentsetting sent to it by the host computer (28) via USB controlcommunication. In USB power mode (in the absence of external power), thecurrent setting output from the DAC (38) is set by the host computer(28) to a value greater than that of the output of the constant voltagereference (36), such that the output of the comparator (37) is high, andsaid output is sent to the AND gate (34) and to the local controller(30). The output of said AND gate (34) is used to control the inputswitch (39) of the current regulator (35). When said output is high, theinput switch (39) connects the current regulator (35) to the output ofthe constant voltage reference (36) derived from USB power, hencesupplying the light source (27).

When external power is supplied to the microscope through the externalpower connector (31), the connection to the local controller (30) causessaid controller to enter external power mode by opening USB SPST powerswitch (32 a) and closing external SPST power switch (32 b), thusconnecting one terminal of the light source (27) to the external powersource, and to signal the status of excitation light source power to thehost computer (28). Said host, in turn, sends the desired current levelfor the current regulator (35) to the DAC (38). In addition, the localcontroller (30) pulls its control output (33) to the AND gate (34) low,such that output of said AND gate sent to the input switch (39) is low.This causes the input switch (39) to connect current regulator (35) tothe DAC (38), thus supplying the excitation light source with externalpower.

In a preferred aspect of the present invention, the host computer (28)can switch the desired current setting of the light source (27) bytoggling its input to DAC (38) between a high value and a low value soas to provide one or more pulses of illumination light, independently ofwhether said light source (27) derives power from USB or an externalsource. These pulses can have durations less than one microsecond andcan have periods elapsing between successive pulses of sufficientduration to enable time-resolved fluorescence from the sample to beacquired by the image sensor.

In a preferred embodiment, the host program provides the ability for theuser to save any image stored in the frame buffer to a file on anarchiving device either present within or attached to the host computer,such as a hard disk drive. In a further specification of the preferredembodiment, the image file may be automatically named with a name thatincludes the date and time at which the image was acquired and stored.In a further aspect of the preferred embodiment, the user may configurethe host program to save multiple acquired images at a user-specifiedperiodic interval, for either a fixed period of time or continuouslyuntil storage is manually stopped by the user, and that the names ofthese sequentially saved files indicate the number of the file in thesequence. When the user-specified interval is zero, the host programarchives each image from the memory buffer in a sequence of contiguousframes analogous to a video stream. An example of the graphical userinterface of the host control program including forms for control ofgain, exposure, and light source power and control of time lapseacquisition and saving of images is shown in FIG. 6.

An x-y caliper can be attached to the stage surface for holdingmicroscope slides, cover slips, and other samples firmly. Motion of thecaliper in each direction may be made to position the sample at preciselocations by the aid of marked vernier distance scales. The caliperenables the sample to be scanned over the field of view to obtain fieldimages separated by accurate distances. The caliper is attached to thestage by knurled screws that can be manually tightened into threadedholes drilled through the upper surface of the stage. This allows thecaliper to be removed from the stage to accommodate large, bulkysamples, such as tissue culture flasks, multiwell plates, and othersamples that are unable to be clamped by the caliper.

Transmission brightfield illumination of the sample is readily achievedwith a light source placed above the observation hole of the stage. In apreferred embodiment, a white LED at the end of a flexible gooseneckconnector is attached to connection 6 (FIG. 1A and FIG. 1B) on the mainunit case. This connector terminates inside the enclosure in a circuitboard-mounted voltage regulator that powers the led. The location anddirection of the brightfield illumination are adjusted manually above oraround the sample to achieve the desired type of illumination. Forexample, acute illumination of the through tissue culture flasks made ofstressed, and thus birefringent plastic slightly polarizes theilluminating light, and enables contrast in the sample to be generatedby interference modulation. In an alternative example, illumination ofthe sample at a highly oblique grazing angle enables an image to thatgenerated by phase-contrast. Transmission white light illumination isuseful for locating regions of the sample to be observed underfluorescence epi-illumination and for obtaining an accurate focusposition.

In a preferred embodiment, the host computer may control bothepi-illumination and brightfield light sources to enable simultaneousacquisition of both brightfield and fluorescence views of the samplewithin the same image acquired by the image sensor. This enables thesource of fluorescence emissions to be localized within a sample towithin non-fluorescent but otherwise refractive or reflective elementsof said sample. In addition, the epi-illumination may be pulsed duringthe acquisition time of an image frame by the image sensor in order todecrease fluorescence noise from the sample recorded by the sensor inthe image.

II. A Method of using an Inverted Fluorescence Microscope

The present invention includes a method of using an invertedfluorescence microscope of the present invention. The method includesproviding a sample and providing a microscope of the present invention.The sample is engaged with the microscope as described herein, and animage is generated. Preferably, there are no structures to allow a humanoperator to view the image directly, such as those structures providedin traditional microscopes. The image is displayed on a screen andoptionally stored on a storage media. The image can be a still shot, assingle frame, a video or a time lapse image. The image can be agrey-scale, a single color, or multiple colors.

EXAMPLES

Because other modifications and changes varied to fit particularoperating requirements and environments will be apparent to thoseskilled in the art, the invention is not considered limited to theexample chosen for purposes of disclosure, and covers all changes andmodifications which do not constitute departures from the true spiritand scope of this invention.

Example 1

This example establishes the ease of use of a microscope of the presentinvention and computer interface and output with a microscope of thepresent invention.

Ease of use of the microscope is shown in FIG. 5A and FIG. 5B, which isa captured screen shot of the host computer running the control programfor the preferred embodiment. An image of human neural stem cellsderived from 13 week-old fetal telencephalon growing in a T25polystyrene tissue culture flask with the growth surface coated withmouse laminins and observed with brightfield illumination through thetop of the flask and with a Meiji 20× magnification 0.4 N.A. 7 mmworking distance objective is shown in the image display area of thegraphical user interface. The image displayed is the last one acquiredduring time lapse acquisition and saving of images once every 10 min.The acquisition sequence had been running continuously for 5 days whenthis screen capture was obtained. The four basic tools for operation ofthe program are located along the bar at the top of the display, andinclude icons (1) for snapping a single image from live, continuousdisplay of sequential images from the sensor, (2) for starting andstopping time-lapse acquisition, (3) for setting up parameters fortime-lapse acquisition, and (4) for user help in operating the program.Under the tool bar is a form for direct reading and writing of controlregisters in the intergrated CMOS sensor; this form can also be closedand removed from the display during normal operation. Control forms forgain, exposure, and illumination power and for entering time-lapseacquisition control parameters, such as the time interval between savedimages, the total period of time during which images are acquired, thefile seed name, and the path to the storage location of the savedimages, are shown overlapping the displayed image. These forms can beclosed or otherwise hidden from view during operation of the microscope.

Example 2

This example establishes the application of a microscope of the presentinvention for educational purposes.

Brightfield images obtained with the educational set of microscopeslides “The 5 Kingdoms” (cat. no. E2-70-4016, Neo/Sci, 80 NorthwestBlvd., Nashua, N.H.) with the use of the Meiji 40× magnification 0.65N.A. 0.5 mm working distance objective in the preferred embodiment isshown in FIG. 7, FIG. 8 and FIG. 9. FIG. 7 is an image of fixedParamecium tetraurelia. Key definitive morphological features of theprotozoans, including cilia and micronuclei, can be resolved. FIG. 8 isan image of a horizontal section through a leaf of Vicia fava (broadbean). The stomata that regulate water exchange between the leaf and theatmosphere and cell nuclei are clearly observed. FIG. 9 is an image ofSpirogyra crassa, a filamentous freshwater green alga. To the left ofthe image, the helical arrangement of photosynthetic chloroplasts aroundprominent cell nuclei is visible in the vertical filament. Extendingacross the image are two filaments undergoing lateral conjugation, withtwo compartments in zygophore stage visible at the right. As “The 5Kingdoms” microscope slide set is aimed to junior high and middle schoolscience education, these examples in FIG. 7, FIG. 8 and FIG. 9demonstrate the capabilities of the preferred embodiment as aneducational tool.

Example 3

This example establishes the application of a microscope of the presentinvention for research and educational purposes.

Usefulness of the preferred embodiment is demonstrated for research andeducation is demonstrated in the fluorescence images of bovine pulmonaryarterial endothelial cells (BPAEC) shown in FIG. 10 and FIG. 11. BPAECwere cultured on a gelatin-coated no. 0 coverslip for 3 days, fixed withbuffered 4% formaldehyde for 15 min, washed three times in buffer,incubated for 3 hrs in BODIPY FL-labeled phallacidin (Life Technologies,Cat. No. B607), washed 3 times in buffer and mounted on a microscopeslide by techniques well-known to those skilled in the art. The slidewas affixed to the x-y stage caliper with tape and mounted on the stageof the preferred embodiment with the coverslip facing the objectivelens. Phallacidin binds to filamentous (F) actin, one of the predominantstructural proteins of eukaryotic cells that maintain their shape andintegrity, and enable their motility.

FIG. 10 was obtained with a Meiji 100× N.A. 1.25 infinity-correctedoil-immersion objective with 0.14 mm working distance. FIG. 11 wasobtained with a Meiji 40× N.A. 0.65 infinity-corrected air objectivewith a 0.5 mm working distance.

The preferred embodiment of the invention was configured forfluorescence as follows: Light from a LUXEON Rebel Blue LED (Part. No.LXML-PB01-0040, Philips Lumileds Lighting Co., San Jose, Calif.) poweredwith a continuous current of 700 mA was passed through a 475 nmcenter-wavelength filter with a full-width at half-maximum (fwhm)passband of 35 nm (Semrock, Inc., Rochester, N.Y.) custom-cut to fit inthe excitation filter position (17) of the optical imager assembly (FIG.2) and the filtered illumination light was reflected to the sample witha 506 nm-edge dichroic beamsplitter (Part. No. FF506-Di03, Semrock) alsocustom-cut to fit at the dichroic mirror position (12). Fluorescenceemitted by the sample was passed through a 535 nm center wavelengthfilter with a fwhm passband of 43 nm (Semrock) custom-cut to fit in theemission filter position (18, see FIG. 2). Projection (tube) lenses usedto focus light collected by the objectives were a 10 mm diameterplanoconvex lens with +20 mm focal length, in tandem with 2 12.5 mm dia.achromat lenses, the first with a 40 mm focal length, and the secondwith a 100 mm focal length. An integrated CMOS sensor (OVT 9715,Omnivision Technologies, Sunnyvale, Calif.) was used to capture theimage.

The 100× oil immersion image of the labeled BPAEC in FIG. 10 revealsconsiderable structural details of the arrangement of F-actin in thesecells. As the depth of field of the 100× objective is so thin (<1 μm),the sample was focused at the cells in the center of the image, suchthat the cells along the bottom of the image, which were at differentheights in the gelatin coating compared to the cells at the center, havebeen allowed to be less well-focused. The nuclear and perinuclear regionis revealed as either brightly labeled, or surrounded by a thick F-actinring. Farther away from the nuclear region, the filaments are lessthick, yet still thicker that the very thin filaments near the peripheryof the cells where the cells extend motile protrusions toward unoccupiedareas of the growth surface.

FIG. 11 was obtained after replacing the 100× objective lens with the40× air objective and reattaching the stage caliper with attachedmicroscope slide. Due to the ˜2 μm depth of field of the lowermagnification objective, the cells throughout the field of view appearto be in focus. The pattern of actin staining observed in greater detailin FIG. 9 is revealed in FIG. 10 to be typical for the cells throughoutthe slide culture. In addition, it is noteworthy that even withmechanical replacement of the objective requiring removal of the rearcover of the preferred embodiment, the center of the field of view inFIG. 10 is at the same location in FIG. 11.

Example 4

This example establishes the application of a microscope of the presentinvention for research purposes.

Further utility of the preferred embodiment for research is illustratedin FIG. 12 and FIG. 13, which were obtained in a setting of collegeinstruction in molecular biology. For this study, transgenic nematodes,Caenorhabditis elegans, were generated by parental transduction with atransposable genetic element encoding Emerald Green Fluorescent Protein(EmGFP) under control of a myosin II promoter element with anintervening nuclear localization signal sequence fused to the N-terminusof the EmGFP. Therefore, the construct is expressed in muscle cells ofthe nematode. FIG. 12 shows an image of the progeny of these transgenicnematodes obtained with a 40× objective in the fluorescenceconfiguration of the preferred embodiment as described in Example 3. Thefluorescence intensity confined to bright oval shapes along the worm arenuclei of muscle cells in the plane of focus, whereas the larger regionsof decreased intensity are nuclei of muscle cells located out of saidplane. FIG. 13 is an image of a comparable nematode progeny of aparental line transduced with the same EmGFP construct, but in additiontransfected with a plasmid bearing a short-hairpin interfering RNA(RNAi). The RNAi sequence was directed to the N-terminus of the EmGFPtranscript, such that transcription of the hairpin resulted in knockdown of EmGFP. The image of the animal in FIG. 13 reveals significantattenuation of nuclear fluorescence compared to that in FIG. 12.

Example 5

This example establishes the application of a microscope of the presentinvention for diagnostic purposes.

Usefulness of the preferred embodiment in diagnostics is illustrated inFIG. 14 and FIG. 15, which are brightfield and fluorescence images,respectively, of a transverse section of the dermal layer of human skincontaining a nevus or mole from a biopsy. The section, obtained andviewed in a dermatology clinic, was stained with hematoxylin and eosinby procedures known to those skilled in the art. FIG. 14 is an image ofthe section under clinic room light, and shows the nevus along theepidermal layer or stratum griseum of the skin with protuberances downinto the dermis. In the image acquired under fluorescence illuminationshown in FIG. 15, collagen and elastin fibers are stained intensely bythe eosin. A clinician uses the relative intensity of this fluorescencestaining to determine the extent to which the nevus has altered thesuppleness of the underlying skin and to judge whether the mole exhibitsdysplasia or is a melanoma warranting excision.

Example 6

This example establishes the application of a microscope of the presentinvention for diagnostic purposes.

Further utility of the preferred embodiment in diagnostics is shown inFIG. 16 and FIG. 17. FIG. 16 shows an unstained section of normal humansmall intestine obtained in fluorescence with the preferred embodiment,showing the intense autofluorescence of the intestinal epithelium liningthe villi consistent with gastrointestinal health. FIG. 17 shows asection from a small intestine tumor obtained from a human patient,showing the profound disorganization of tissue fluorescencecharacteristic of tumor growth.

Example 7

This example establishes the application of a microscope of the presentinvention for research purposes when multiple fluorescent dyes arepresent in the sample.

For this example, a specimen is labeled with multiple fluorescent dyes,each of which is maximally excited to fluorescence emission at adifferent excitation wavelength, and each of which emits maximally adifferent wavelength of light. Such multiple dye labeling is well-knownto those skilled in the art, and may be obtained by indirectimmunofluorescence of different epitopes in a sample with primaryantibodies raised against said epitopes in different mammalian species,followed by binding isotype-matched secondary antibodies with eachsecondary antibody labeled with a different fluorescent dye.

A microscope of the present invention is configured for fluorescence asfollows: Light from a LUXEON Rebel Blue LED (Part. No. LXML-PB01-0040,Philips Lumileds Lighting Co., San Jose, Calif.) powered with acontinuous current of 700 mA is passed through an excitation filterhaving at least two center-wavelengths with non-overlapping fwhmpassbands sufficiently narrow such that at least two wavelengths ofwell-separated light are produced. This filter is placed in theexcitation filter position (17) of the optical imager assembly (FIG. 2).The filtered illumination light is reflected to the sample with adichroic beamsplitter located at the dichroic mirror position (12). Thedichroic beamsplitter is selected so that the edge wavelength reflectsthe wavelengths of excitation light. Fluorescence emitted by the sampleis passed through an emission filter located at the emission filterposition (18, see FIG. 2) having center-wavelengths matched to thewavelengths emitted by the multiple fluorescent dyes. The fwhm passbandssurrounding these center wavelengths are chosen such that each dye'sfluorescence emission is well-separated from the other dyes. Projection(tube) lenses are used focus light collected by the objective lens ontoan integrated CMOS sensor (OVT 9715, Omnivision Technologies, Sunnyvale,Calif.) is used to capture the image. Fluorescence by the multiple dyesis separated into separate images, one for each dye, by the hostcomputer applying a selection algorithm to the Bayer color pattern ofthe resulting image.

Example 8

This example establishes the application of a microscope of the presentinvention for research purposes for acquiring one or more simultaneousbrightfield and fluorescence images in which fluorescence noise isdecreased by pulsing the excitation light source.

The sample is illuminated by the brightfield light source. The hostcomputer program then pulses the epi-illumination light source with asignal less than 10 μsec in duration with a delay allowing read out ofimage data from the sensor to be triggered at a user-specified delay.The resulting image shows fluorescence overlayed on a brightfield imageof the sample.

Example 9

This example establishes the application of a microscope of the presentinvention for research purposes by using time-lapse acquisition ofmultiple images of a dynamic biological process.

The glass bottom (0.15 mm thin) of a FluoroDish (Cat. No. FD-35, WorldPrecision Instruments, Ltd., Hertforshire, UK) was coated with 10 μg/mlPoly-L-Ornithine in water for 24 hr at 37° C. After washing withphosphate-buffered saline, the glass surface was coated with 10 μg/mlmouse laminins in water for 24 hr. The surface was seeded with theneural stem cells described in Example 1 at a density of 100,000 cellsper cm². A microscope of the present invention was placed in ahumidified incubator maintained continuously at a temperature of 37° C.,and the covered FluoroDish was placed on the stage. The sample wasobserved with a 40× objective. The program on the host computer wasconfigured to illuminate the brightfield LED for 4 sec once every 10min, during which time an image was acquired from the sensor andarchived with a file name containing indicia of the date and time thatthe image was acquired. FIG. 18 is an image obtained after the fetalneural stem cells had settled on the laminin-coated glass surface within6 hr of seeding. FIG. 19 is an image acquired 5 days later, revealingthe expression of extensive lamellipodia exploring the laminin-coatedsurface, and the formation of proliferation colonies.

REFERENCES

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1. An inverted fluorescence microscope, comprising: a. at least onestage for placing at least one sample for observation; b. a at least oneepifluorescence illumination and detection system, comprising: i. atleast one filter set below said stage, comprising: a) at least oneexcitation filter; b) at least one dichroic mirror; and c) at least oneemission filter; ii. at least one objective lens between said stage andsaid filter set; iii. at least one image sensor below said filter set;iv. at least one epi-illumination system below said stage, comprising:a) at least one source of epi-illumination light, b) at least onecondenser lens; and c) at least one said excitation filter of saidfilter set, v. at least one imaging barrel below said dichroic mirror insaid filter set, comprising: a) said emission filter; b) at least oneprojection lens; and c) said image sensor; c. at least one focusingassembly that attaches said epi-fluorescence illumination and detectionsystem to said stage; and d. at least one host computer comprising atleast one program to control image acquisition of at least one imagefrom said image sensor.
 2. The inverted fluorescence microscope of claim1, wherein said stage comprises an x-y clamping caliper system forhorizontal movement of said sample.
 3. The inverted fluorescencemicroscope of claim 1, wherein said epifluorescence illumination anddetection system lacks a direct optical human interface.
 4. The invertedfluorescence microscope of claim 1, further comprising at least onesample cover to shield said stage from an outside light source or astray light source.
 5. The inverted fluorescence microscope of claim 4,wherein said sample cover can be displaced to expose said stage.
 6. Theinverted fluorescence microscope of claim 1, wherein saidepi-illumination system extends in a generally horizontal direction fromsaid filter set.
 7. The inverted fluorescence microscope of claim 1,wherein said excitation filter provides non-vergent or vergent focus ofsaid source of epi-illumination light on said sample.
 8. The invertedfluorescence microscope of claim 1, wherein in said epifluorescenceillumination and detection system, said source of epi-illuminationlight, said condenser lens, and said excitation filter are mounted in atleast one illumination tube as a generally horizontal extension from atleast one filter cube with at least one optical axis extendinghorizontally toward said filter set from said source of epi-illuminationlight, and light emitted from said source of epi-illumination lightbeing directed to said stage by way of said dichroic mirror.
 9. Theinverted fluorescence microscope of claim 8, wherein said filter cubecomprises an about 45 degree slanted end of said illumination tube, andcomprises said dichroic mirror centered at an optical axis extendingfrom said objective lens to said projection lens and focuses at leastone sample image on said image sensor, wherein along said optical axissaid emission filter is provided between said dichroic mirror and saidprojection lens.
 10. The inverted fluorescence microscope of claim 1,wherein said imaging barrel focuses at least one filtered image ontosaid image sensor.
 11. The inverted fluorescence microscope of claim 1,wherein said imaging barrel is mounted in at least one tube extending ina generally vertical direction away from said filter set and focuses atleast one filtered image of said sample onto said image sensor.
 12. Theinverted fluorescence microscope of claim 1, wherein in said focusingassembly, the field of view does not substantially shift during focusingand that variation of focus due to mechanical vibration is minimized.13. The inverted fluorescence microscope of claim 1, wherein said sourceof epi-illumination light comprises at least one light-emitting diode(LED).
 14. The inverted fluorescence microscope of claim 1, wherein saidsource of epi-illumination light comprises at least one laser diode. 15.The inverted fluorescence microscope of claim 1, wherein said programcontrols at least one image display from said image sensor.
 16. Theinverted fluorescence microscope of claim 1, wherein said programconfigures at least one image display from said image sensor.
 17. Theinverted fluorescence microscope of claim 1, wherein said image sensordetects multiple fluorescent emissions of differing wavelengths.
 18. Theinverted fluorescence microscope of claim 17, wherein said multiplefluorescent emissions are from different fluorescent labels in saidsample.
 19. The inverted fluorescence microscope of claim 1, whereinsaid excitation filter provides a plurality of wavelengths or range ofwavelengths from said source of epi-illumination light.
 20. The invertedfluorescence microscope of claim 1, wherein said dichroic mirrorprovides excitation light towards said sample and transmits emissionlight towards said image sensor. 21-31. (canceled)